CN109668980A - A kind of vitamin K2(MK-7) detection method of content - Google Patents
A kind of vitamin K2(MK-7) detection method of content Download PDFInfo
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
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Abstract
The invention discloses a kind of vitamin Ks2(MK-7) detection method of content, belong to compounds content detection technique field, it comprises the following processes: A. sample pretreatment, B. reference substance solution is prepared, C. the preparation of solution to be measured, D. standard reference material solution is measured using high-efficient liquid phase chromatogram technology, E. is measured testing sample solution using high-efficient liquid phase chromatogram technology, and F. calculates vitamin K in sample to be tested according to external standard method2(MK-7) content C.Detection method of the invention can improve functional component recovery rate, and testing result is accurate and reliable, and detection method is easy, efficient；Detection method of the invention has wide range of applications, and can be used for the content quick determination containing menaquinone (MK-7) health food, drug, ordinary food or premix etc..
Description
Technical field
The present invention relates to health food or drug detection fields, more particularly to contain vitamin K2Soft capsule (MK-7),
The detection method of menaquinone functional component content in the compound formulations such as tablet, granule.
Background technique
Vitamin K2, also known as menadione is light yellow crystal, mainly synthesized by enteric bacteria；It is by a methyl naphthalene
Quinone female ring and one kind of an isoprene side chains composition being located on the position female ring C-3 have the fat-soluble chemical combination of quinones structure
Object.Different according to isoprene unit number on side chain, menadione is divided into 14 kinds, is indicated with MK-n, has heat resistance, but right
Light and alkali are sensitive.Menaquinone (MK-7) is a member in MK-n, contains 7 isoprene units on side chain.
Menaquinone (MK-7) has longer half-life period, very stable in blood, thus is that mammal generation is solidifying
A very important co-factor of blood factor II, VII, IX and X, are indispensable nutrients to blood clotting, there is research
Report, MK-7 have the function of blood pressure lowering.In addition, MK-7 can inhibit the growth of osteoblast, to stimulate osteoblast
Differentiation forms bone.There is foreign scholar to point out, mine in the elderly's bone can be significantly improved by supplementing a certain amount of MK-7 daily
Material density (BMD) reduces the hip fracture risk of the elderly, and supplements on a small quantity in Pre-adolescence children's body of health
MK-7 content free in vivo can be improved in MK-7, is conducive to the carboxylation for improving osteocalcin.In addition, MK-7 can assist in
Regulate and control Ca2+Utilization and promote Gla protein (Matrix Gla Protein, MGP) activity, to prevent calcareous in blood vessel
It is middle precipitating and caused by artery sclerosis.Therefore, MK-7 is the health care that the third generation emerging in the world prevents and treats osteoporosis
Food, safety have obtained the power that U.S. FDA, China SFDA, United States drug study European Parliament and EU Council in one's power
Prestige identification, function are receiving more and more attention.
Currently, seldom being reported for menaquinone (MK-7) assay in health food, especially in compound formulation
Menaquinone (MK-7) assay." vitamin K in rp-hplc determination natto extract2(35)
Content " (" the Chinese drug standards " the 4th phase page 287 of volume 11 in 2010) sample treatment and chromatographic condition reported in the literature
It cannot effectively extract menaquinone micro in compound formulation (MK-7), and cannot be effectively by purpose peak and impurity peaks point
From；The present inventor using the method in above-mentioned document to vitamin K soft capsule, calcium Vitamin D Vitamin K soft capsule and
Calcium Vitamin D Vitamin K chewable tablets etc. carries out vitamin K2(MK-7) measurement of content, measurement result show vitamin K2(MK-
7) separating degree of main peak cannot effectively separate at purpose peak with impurity peaks less than 1.5.A kind of " vitamin K2(MK-7) content
Measuring method " vitamin K disclosed in (CN104458990)2(MK-7) detection method of content is extracted from different calcium preparations
Active constituent content is relatively low, and sample sample weighting amount has certain influence relative to constant volume, and the not easy to operate and processing time is longer；
The present inventor is using the above method to calcium Vitamin D Vitamin K soft capsule and calcium Vitamin D Vitamin K chewable tablets etc.
Carry out vitamin K2(MK-7) measurement of content, measurement result shows that the recovery rate of effective component is extremely low, and then causes to determine
Vitamin K2(MK-7) content is lower than actual content 50% even more；In addition, in the sample to be tested of this method preparation
Effective component is lower, for using soybean oil, corn oil or other plants in compound formulation such as soft capsule containing more auxiliary material
Object oil, cannot effectively separate impurity peaks in auxiliary material with purpose peak.
Summary of the invention
The technical problem to be solved by the invention is to provide a kind of vitamin Ks2(MK-7) detection method of content improves function
The recovery rate for imitating ingredient, improves the accuracy of testing result.
In order to solve the above technical problems, the technical scheme adopted by the invention is that:
A kind of vitamin K2(MK-7) detection method of content, comprises the following processes:
A. sample pretreatment: 1. weighing quality is MSampleSample to be tested be placed in reaction vessel, be added mixed solution, mixing
Ultrasonic treatment, is then centrifuged for taking supernatant solution after uniformly；2. acid solution is added into supernatant solution, sufficiently after oscillation, will be layered
Lower layer's solution removal afterwards；3. water is added into step 2. remaining solution, sufficiently after oscillation, lower layer's solution after layering is gone
It removes；4. collection step 3. upper solution, is spin-dried for, it is V that organic solvent constant volume, which is added, and obtains volumeSampleSample solution；
B. it prepares reference substance solution: weighing vitamin K2(MK-7) reference substance obtains concentration after organic solvent constant volume is added
For CIt is rightReference substance solution；
C. the preparation of solution to be measured: sample solution, reference substance solution are filtered through miillpore filter respectively, are obtained to be measured
Sample solution, standard reference material solution；
D. standard reference material solution is measured using high-efficient liquid phase chromatogram technology, obtains vitamin K2(MK-7) chromatography
The peak area at peak is simultaneously denoted as AIt is right, sampling volume is denoted as VIt is right；
E. it utilizes identical high-efficient liquid phase chromatogram technology condition in same step D to be measured testing sample solution, obtains
Vitamin K2(MK-7) peak area of chromatographic peak and it is denoted as ASample, sampling volume is denoted as VLoading；
F. vitamin K in sample to be tested is calculated according to external standard method2(MK-7) content C, calculation formula are as follows:
The unit of C is μ g/g, V in formulaSampleUnit be mL, CIt is rightUnit be μ g/mL, VIt is right、VLoadingUnit be μ L, MSample's
Unit is g.
Technical solution of the present invention further improvement lies in that: the sample to be tested be solid when, be first ground into fine powder again into
Row pretreatment；Sample to be tested is directly pre-processed when being liquid.
Technical solution of the present invention further improvement lies in that: the mixed solution is mixed by polar solvent and nonpolar solvent
It forms.
Technical solution of the present invention further improvement lies in that: the polar solvent be isopropanol, methanol, ethyl alcohol, in acetonitrile
It is any；Nonpolar solvent is any one of n-hexane, petroleum ether, hexamethylene.
Technical solution of the present invention further improvement lies in that: the mixed solution is mixed by n-hexane and isopropanol.
Technical solution of the present invention further improvement lies in that: the volume ratio of the n-hexane and isopropanol be 2~4:1.
Technical solution of the present invention further improvement lies in that: the acid is glacial acetic acid, and the concentration of acid solution is 2~4mol/
L。
Technical solution of the present invention further improvement lies in that: organic solvent described in step A and step B be methanol, ethyl alcohol,
Any one of isopropanol or acetonitrile, and organic solvent type used is identical in step A and step B.
Technical solution of the present invention further improvement lies in that the condition of the high performance liquid chromatography are as follows: chromatographic column is with octadecane
Base silane bonded silica gel is filler or equivalent chromatographic column；Mobile phase include acetonitrile and methanol solution, flow rate of mobile phase be 0.8~
1.5mL/min, column temperature are 25~40 DEG C, and sample volume is 10~20 μ L, and Detection wavelength is UV, visible light 254nm.
Technical solution of the present invention further improvement lies in that: described is gradient elution when being eluted with mobile phase, is specially existed
The content of acetonitrile is 70~80% in 0~10min mobile phase, the content of methanol is 20~30%；It is flowed in 10~20min
The content that the content of acetonitrile is at the uniform velocity reduced to 0, methanol by 70~80% in phase at the uniform velocity rises to 70~80% by 20~30%；The 20th
~50min mobile phase is methanol.
By adopting the above-described technical solution, the technological progress achieved by the present invention is:
Detection method of the invention can improve functional component recovery rate, the impurity being effectively introduced into auxiliary material in sample to be tested
Peak is separated with purpose peak, and testing result is accurate and reliable, and detection method is easy, efficient；Detection method of the invention has a wide range of application
It is general, it can be used for the content quick determination containing menaquinone (MK-7) health food, drug, ordinary food or premix etc..
Preprocessing process in detection method makes vitamin K in compound formulation2(MK-7) it is utmostly mentioned
It takes out, to improve the detectable concentration of functional component, and finally improves the accuracy and reliability of testing result.
Method in detection method when high performance liquid chromatography detection using gradient elution, so that impurity peaks and purpose
Peak, which is kept completely separate, to come, and improves the accuracy of effective substance peak value, further improves the accuracy of testing result.
Detection method of the invention is applied widely, can be used for containing the plurality kinds of health care such as multivitamin and calcium preparation
Menaquinone assay in food, drug or food.
Detailed description of the invention
Fig. 1 is vitamin K in embodiment 12(MK-7) reference substance map；
Fig. 2 is the HPLC-UV of sample solution in embodiment 1；
Fig. 3 is vitamin K in embodiment 22(MK-7) reference substance map；
Fig. 4 is the HPLC-UV of sample solution in embodiment 2；
Fig. 5 is vitamin K in embodiment 32(MK-7) reference substance map；
Fig. 6 is the HPLC-UV of sample solution in embodiment 3；
Fig. 7 is vitamin K2Content linear relationship chart；
Fig. 8 is vitamin K soft capsule blank sample chromatogram；
Fig. 9 is vitamin K calcium vitamin D soft capsule blank sample chromatogram；
Figure 10 is vitamin K calcium vitamin D chewable tablets blank sample chromatogram；
Figure 11 is vitamin K under vitamin K soft capsule testing conditions2Reference substance solution chromatogram；
Figure 12 is vitamin K under vitamin K calcium vitamin D soft capsule testing conditions2Reference substance solution chromatogram；
Figure 13 is vitamin K under vitamin K calcium vitamin D chewable tablets testing conditions2Reference substance solution chromatogram；
Figure 14 is the chromatogram that testing sample solution carries out high-efficient liquid phase color time spectrum in comparative example 1；
Figure 15 is the chromatogram that testing sample solution carries out high-efficient liquid phase color time spectrum in comparative example 2；
Figure 16 is the chromatogram that testing sample solution carries out high-efficient liquid phase color time spectrum in comparative example 3；
Figure 17 is the chromatogram that testing sample solution carries out high-efficient liquid phase color time spectrum in comparative example 4.
Specific embodiment
The present invention is described in further details below with reference to embodiment:
Embodiment 1
1. sample to be tested
1.1 vitamin K soft capsules: lot number: VK180001, production unit: Handan Co., Ltd, morning twilight biotechnology group
2. reagent and material
2.1 isopropanols (analysis is pure)
2.2 petroleum ethers (analysis is pure)
2.3 methanol (analysis is pure)
2.3 acetonitriles (chromatographically pure)
2.4 methanol (chromatographically pure)
3. chromatographic condition
3.1 chromatographic columns: Amethyst C18-H (4.6 × 150mm, 5 μm)；
3.2 mobile phases: using acetonitrile as mobile phase A, using methanol as Mobile phase B, gradient is carried out according to the regulation in following table and is washed
It is de-；
3.3 flow rate of mobile phase: 0.8mL/min；
3.4 column temperatures: 40 DEG C；
3.5 sample volumes: 10 μ L；
3.6 Detection wavelengths: UV, visible light 254nm；
4. analytical procedure
4.1 prepare reference substance solution: precision weighs 10mg vitamin K2(MK-7) reference substance is in 100mL brown volumetric flask
In, 50mL isopropanol is added, ultrasound dissolves reference substance sufficiently, isopropanol is added to be settled to graduation mark, as stock solution.Precision amount
It takes reference substance stock solution 2mL in 10mL brown volumetric flask, 20.0 μ g/mL (C is madeIt is right) reference substance solution.
4.2 accurately weigh sample to be tested 1.5258g (MSample) be placed in 20mL brown volumetric flask, be added 7.5mL petroleum ether and
The methanol mixed solution that 3:1 is formed by volume, oscillation are uniformly mixed, ultrasonic 30min, are then centrifuged for (5000r/min centrifugation
10min) take supernatant solution；The glacial acetic acid solution that concentration is 2mol/L is added into supernatant solution will be layered sufficiently after oscillation
Lower layer's solution removal afterwards；Water is added into above-mentioned remaining solution, sufficiently after oscillation, lower layer's solution after layering is removed；
Remaining solution is spin-dried for, is cooled to room temperature, isopropanol constant volume is added, shakes up to get sample solution, volume is denoted as VSample。
4.3 filtering with microporous membrane by the testing sample solution of reference substance solution and step 1 through 0.45 μm (or 0.22 μm),
Standard reference material solution and testing sample solution are obtained, two kinds of solution are subjected to reversed efficient liquid phase chromatographic analysis, 10 μ L of sample introduction.
4.4 assays: vitamin K in sample is calculated according to following equation2(MK-7) content.
A in formulaSample: the vitamin K of testing sample solution2Peak area；
VSample: testing sample solution total volume, mL；
CIt is right: the concentration of reference substance solution, μ g/ml；
VIt is right: the sampling volume of reference substance solution, μ L；
AIt is right: the vitamin K of reference substance solution2(MK-7) peak area；
VLoading: testing sample solution loading volume, μ L；
MSample: sample to be tested sample weighting amount, g.
In the present embodiment, reference substance solution carries out high performance liquid chromatography measurement, with qualitative (the chromatogram such as Fig. 1 of retention time
It is shown), to obtain vitamin K2(MK-7) peak area of chromatographic peak；And it in conjunction with the sample weighting amount of sample to be tested, obtains to be measured
Concentration quantitative in sample solution (chromatogram is as shown in Figure 2).Calculate vitamin K2(MK-7) content is 340 μ g/
g。
Embodiment 2
1. sample to be tested
1.1 calcium Vitamin D Vitamin K soft capsules, lot number: KGR180001, production unit: morning twilight biotechnology group Handan
Dancheng Co., Ltd
2. reagent and material
2.1 n-hexanes (analysis is pure)
2.2 isopropanols (analysis is pure)
2.3 petroleum ethers (analysis is pure)
2.4 acetonitriles (chromatographically pure)
2.5 methanol (chromatographically pure)
3. chromatographic condition
3.1 chromatographic columns: SPOLAR C18 (4.6 × 250mm, 5 μm)；
3.2 mobile phases: using acetonitrile as mobile phase A, using methanol as Mobile phase B, gradient is carried out according to the regulation in following table and is washed
It is de-；
3.3 flow rate of mobile phase: 1.0mL/min；
3.4 column temperatures: 30 DEG C；
3.5 sample volumes: 10 μ L of reference substance solution sample introduction, 20 μ L of sample to be tested sample introduction；
3.6 Detection wavelength: UV, visible light 254nm；
4. analytical procedure
4.1 prepare reference substance solution: precision weighs 10mg vitamin K2(MK-7) reference substance is in 100mL brown volumetric flask
In, 50mL isopropanol is added, ultrasound dissolves reference substance sufficiently, isopropanol is added to be settled to graduation mark, as stock solution.Precision amount
It takes reference substance stock solution 2mL in 10mL brown volumetric flask, 20.0 μ g/mL (C is madeIt is right) reference substance solution.
4.2 accurately weigh sample to be tested 6.1071g (MSample) be placed in stuffed conical flask, 30mL n-hexane and isopropanol is added
(2:1) mixed solution, oscillation are uniformly mixed, and ultrasonic 40min, 5000r/min are centrifuged 10min, take supernatant solution that 50mL is added dense
Degree is the glacial acetic acid solution of 3mol/L, sufficiently vibrates, removes lower layer's solution after layering, water is added in remaining solution, sufficiently
Oscillation, then lower layer's solution is removed after secondary clearing, it takes upper solution to be spin-dried for, isopropanol is added and is settled to 20mL, shakes up to get sample
Solution, volume are denoted as VSample。
4.3 filtering with microporous membrane by the sample solution of reference substance solution and step 1 through 0.45 μm (or 0.22 μm) obtains
Standard reference material solution and testing sample solution carry out reversed efficient liquid phase chromatographic analysis, 10 μ L (V of reference substance solution sample introductionIt is right),
20 μ L (V of testing sample solution sample introductionLoading)。
4.4 assays: vitamin K in sample is calculated according to following equation2(MK-7) content.
A in formulaSample: the vitamin K of testing sample solution2Peak area；
VSample: testing sample solution total volume, mL；
CIt is right: the concentration of reference substance solution, μ g/ml；
VIt is right: the sampling volume of reference substance solution, μ L；
AIt is right: the vitamin K of reference substance solution2(MK-7) peak area；
VLoading: testing sample solution loading volume, μ L；
MSample: sample to be tested sample weighting amount, g.
In the present embodiment, reference substance solution carries out high performance liquid chromatography measurement, with qualitative (the chromatogram such as Fig. 3 of retention time
It is shown), to obtain vitamin K2(MK-7) peak area of chromatographic peak；And it in conjunction with the sample weighting amount of sample to be tested, obtains to be measured
Concentration quantitative in sample solution (chromatogram is as shown in Figure 4).Calculate vitamin K2(MK-7) content is 41.25 μ
g/g。
Embodiment 3
1. sample to be tested
1.1 calcium Vitamin D Vitamin K chewable tablets, lot number: KGJ180002, production unit: morning twilight biotechnology group Handan
Dancheng Co., Ltd
2. reagent and material
2.1 isopropanols (analysis is pure)
2.2 hexamethylenes (analysis is pure)
2.3 acetonitriles (analysis is pure)
2.4 acetonitriles (chromatographically pure)
2.5 dehydrated alcohols (chromatographically pure)
3. chromatographic condition
3.1 chromatographic columns: Shim-pack GIST C18 (4.6 × 250mm, 5 μm)；
3.2 mobile phases: using acetonitrile as mobile phase A, using methanol as Mobile phase B, gradient is carried out according to the regulation in following table and is washed
It is de-；
3.3 flow rate of mobile phase: 1.5mL/min；
3.4 column temperatures: 25 DEG C；
3.5 sample volumes: 10 μ L of reference substance solution sample introduction, 20 μ L of sample to be tested sample introduction；
3.6 Detection wavelengths: UV, visible light 254nm；
4. analytical procedure
4.1 prepare reference substance solution: precision weighs 20mg vitamin K2(MK-7) reference substance is in 100mL brown volumetric flask
In, 50mL isopropanol is added, ultrasound dissolves reference substance sufficiently, isopropanol is added to be settled to graduation mark, as stock solution.Precision amount
It takes reference substance stock solution 2mL in 10mL brown volumetric flask, the reference substance solution of 40.0 μ g/mL is made.
If 4.2 randomly select dry plate sample, it is ground into fine powder, accurately weighs 10.0679g, then be placed in stuffed conical flask,
30mL hexamethylene and acetonitrile (4:1) mixed solution is added, oscillation is uniformly mixed, and ultrasonic 40min, 5000r/min are centrifuged 10min,
It takes supernatant solution that 30mL glacial acetic acid solution is added, sufficiently vibrates, lower layer's solution is removed after layering, water is then added, sufficiently shake
It swings, then removes lower layer's solution after secondary clearing, upper solution is taken to be spin-dried for, isopropanol is added and is settled to 20mL, shakes up to get sample
Solution.
4.3 filtering with microporous membrane by the sample solution of reference substance solution and step 1 through 0.45 μm (or 0.22 μm) obtains
Standard reference material solution and testing sample solution, carry out reversed efficient liquid phase chromatographic analysis, reference substance solution sample introduction 10 μ L, to be measured
20 μ L of sample solution sample introduction.
4.4 assays: vitamin K in sample is calculated according to following equation2(MK-7) content.
A in formulaSample: the vitamin K of testing sample solution2Peak area；
VSample: testing sample solution total volume, mL；
CIt is right: the concentration of reference substance solution, μ g/ml；
VIt is right: the sampling volume of reference substance solution, μ L；
AIt is right: the vitamin K of reference substance solution2(MK-7) peak area；
VLoading: testing sample solution loading volume, μ L；
MSample: sample to be tested sample weighting amount, g.
In the present embodiment, reference substance solution carries out high performance liquid chromatography measurement, with qualitative (the chromatogram such as Fig. 5 of retention time
It is shown), to obtain vitamin K2(MK-7) peak area of chromatographic peak；And it in conjunction with the sample weighting amount of sample to be tested, obtains to be measured
Concentration quantitative in sample solution (chromatogram is as shown in Figure 6).Calculate vitamin K2(MK-7) content is 38.92 μ
g/g。
Embodiment 4-6
Embodiment 4-5 and 2 step of embodiment and parameter are essentially identical, and distinctive points are the mixed of sample dissolution in embodiment 4
Closing solution is the mixed solution that petroleum ether and methanol form, and isopropanol used in constant volume is replaced with methanol；Sample dissolution in embodiment 5
Mixed solution be mixed solution that hexamethylene and acetonitrile form, isopropanol used in constant volume is replaced with acetonitrile, remaining parameter and reality
It is identical to apply example 2；Embodiment 6 and 3 step of embodiment and parameter are essentially identical, and distinctive points are the mixed of sample dissolution in embodiment 6
Closing solution is the mixed solution that petroleum ether and isopropanol form, and isopropanol used in constant volume is replaced with ethyl alcohol, remaining parameter and implementation
Example 3 is identical.
Testing result are as follows: embodiment 4 is 41.22 μ g/g, and embodiment 5 is 41.27 μ g/g, with 2 testing result base of embodiment
This is identical；Embodiment 6 is 38.96 μ g/g, essentially identical with testing result in embodiment 3.
It is following to be verified for detection method
Chromatographic condition and system suitability
Chromatographic column: using octadecylsilane chemically bonded silica as filler (4.6 × 250mm, 5 μm of partial size)
Mobile phase:
Using acetonitrile as mobile phase A, using methanol as Mobile phase B, gradient elution is carried out according to the regulation in following table；
Flow rate of mobile phase: 1.0mL/min；
Column temperature: 30 DEG C；
Detection wavelength: UV, visible light 254nm；
1) system suitability
Precision weighs 10mg vitamin K2(MK-7) 50mL isopropanol is added in 100mL brown volumetric flask in reference substance, surpasses
Sound dissolves reference substance sufficiently, and isopropanol is added to be settled to graduation mark, as stock solution.Precision measure reference substance stock solution 2mL in
In 10mL brown volumetric flask, the reference substance solution of 20.0 μ g/mL is made.
The above-mentioned 10 μ L of reference substance solution of precision measurement, injection liquid chromatograph, record chromatogram, 6 needle of continuous sample introduction, as a result
It is shown in Table 1.
1 reference substance solution system suitability measurement result of table
Test result shows: vitamin K in 6 needle reference substance solutions2The RSD of peak peak area is less than 2.0%；
2) linear relationship and range
Take vitamin K2Reference substance stock solution is added isopropanol and is formulated as follows concentration: 2.0 μ g/mL, 5.0 μ g/mL, 10.0 μ
G/mL, 20.0 μ g/mL, 40.0 μ g/mL, 60.0 μ g/mL, precision measure above-mentioned each 10 μ L of linear relationship solution, are injected separately into liquid
Chromatography records chromatogram.Linear regression is carried out to peak area with each component concentration in linear relationship solution, obtains linearly side
Journey.It the results are shown in Table 2 and Fig. 7.
2 vitamin K of table2Linear test result
| Concentration (μ g/mL) | 2 | 5 | 10 | 20 | 40 | 60 |
| Peak area | 28.6 | 70.1 | 140.3 | 280.1 | 558 | 845 |
Test result shows: vitamin K2It is linear, linear equation phase within the scope of 2.0 μ of μ g/mL~60.0 g/mL in concentration
Close coefficients R2Greater than 0.999, meet assay requirement.
3) accuracy test
Precision weighs 10mg vitamin K2(MK-7) 50mL isopropanol is added in 100mL brown volumetric flask in reference substance, surpasses
Sound dissolves reference substance sufficiently, and isopropanol is added to be settled to graduation mark, as stock solution.Appropriate reference substance stock solution is pipetted, to calcium
0.8 times, 1.0 times, 1.2 times of " sample " ingredients to be measured are added in Vitamin D Vitamin K soft capsule (lot number: KGR180001) sample
The reference substance (each 3 parts of parallel sample) of contents level, respectively accuracy solution 1, accuracy solution 2, accuracy solution 3 (add
Scalar be equivalent to content 80%, 100%, 120%), precision measures above-mentioned each 20 μ L of solution, be injected separately into liquid chromatograph,
Record chromatogram.The rate of recovery is calculated by peak area external standard method, the results are shown in Table 3.
3 vitamin K of table2Content accuracy test result
Test result shows: under three concentration, vitamin K2The rate of recovery of content is between 98~102%；It is same
Under concentration, the RSD value of determination of recovery rates result is respectively less than 2% three times.This law accuracy is good, meets assay requirement.
4) repeatability and measurement range
Testing sample solution: calcium Vitamin D Vitamin K soft capsule (lot number: KGR180002), accurately weighed, test specimens are taken
Product weight ± 20% is investigated, and prepares 3 parts of sample to be tested concentration respectively.
Reference substance solution: precision weighs 10mg vitamin K2(MK-7) reference substance is added in 100mL brown volumetric flask
50mL isopropanol, ultrasound dissolve reference substance sufficiently, and isopropanol is added to be settled to graduation mark, as stock solution.Precision measures control
The reference substance solution of 20.0 μ g/mL is made in 10mL brown volumetric flask in product stock solution 2mL.
Precision measures 10 μ L of reference substance solution and 20 μ L of testing sample solution, is injected separately into liquid chromatograph, records chromatography
Figure calculates vitamin K in testing sample solution by external standard method2Content the results are shown in Table 4.
4 vitamin K of table2Content repetitive test result
Test result shows: the vitamin K that sample to be tested measures2The RSD of average content is less than 2.0%.This method repeats
Property is good, meets assay requirement.
5) specificity is investigated
It is buckled respectively in vitamin K soft capsule, calcium Vitamin D Vitamin K soft capsule and calcium Vitamin D Vitamin K chewable tablets
Except vitamin K2Raw material, other supplementary material ingredient configuration proportions are constant, and simulation blank sample carries out the investigation of method specificity, blank
Sample, vitamin K2Reference substance liquid chromatogram is shown in Fig. 8,9,10,11,12,13 respectively.
In the liquid chromatogram of blank sample solution, in vitamin K2It is not interfered at reference substance appearance time, without miscellaneous
Matter interference；Vitamin K2In the liquid chromatogram of reference substance solution, vitamin K2It is well-symbolized, tailing factor 0.997；To
In the liquid chromatogram of sample solution, vitamin K2Separating degree is greater than 1.5, meets measurement and requires.
Comparative example 1
Comparative example 1 is the comparative test of embodiment 1, and distinctive points are that the preprocessing process of sample in comparative example 1 is to weigh
Then sample to be tested is directly added into isopropanol, ultrasound dissolves sample sufficiently, and isopropanol constant volume is added and obtains sample solution,
Remaining processing step and parameter are same as Example 1, and the detection of comparative example 1 obtains vitamin K in sample2(MK-7) content is
It is low to detect numerical value than embodiment 1 by 290 μ g/g.Figure 14 is the chromatogram that testing sample solution carries out high-efficient liquid phase color time spectrum.
Comparative example 2
Comparative example 2 is the comparative test of embodiment 2, and distinctive points are that the preprocessing process of sample in comparative example 2 is to weigh
Then sample to be tested is directly added into isopropanol, ultrasound dissolves sample sufficiently, and isopropanol constant volume is added and obtains sample solution,
Remaining processing step and parameter are same as Example 2, and the detection of comparative example 2 obtains vitamin K in sample2(MK-7) content is
It is much lower to detect numerical value than embodiment 2 by 25.13 μ g/g.Figure 15 is the chromatography that testing sample solution carries out high-efficient liquid phase color time spectrum
Figure.
Comparative example 3
Comparative example 3 is the comparative test of embodiment 2, and distinctive points are that the preprocessing process of sample in comparative example 2 is to weigh
Then sample to be tested is directly added into isopropanol, ultrasound dissolves sample sufficiently, and isopropanol constant volume is added and obtains sample solution,
Mobile phase uses dehydrated alcohol when high performance liquid chromatography detects: the acetonitrile mobile phase that 55::45 is formed by volume is washed
De-, remaining processing step and parameter are same as Example 2, and the detection of comparative example 2 obtains vitamin K in sample chromatogram figure2(MK-7)
The separating degree of main peak can not be efficiently separated less than 1.5 with impurity peaks.Figure 16 is that testing sample solution carries out high-efficient liquid phase color time spectrum
Chromatogram.
Comparative example 4
Comparative example 4 is the comparative test of embodiment 3, and distinctive points are that the preprocessing process of sample in comparative example 4 is to weigh
Sample to be tested, is then added the accurate isopropanol for measuring certain volume, and 55 DEG C of thermostat water bath vortex oscillation 40min fill sample
Point dissolution is to get sample solution is arrived, and mobile phase uses methanol to be eluted for mobile phase when high performance liquid chromatography detection, remaining work
Skill step and parameter are same as Example 3, and the detection of comparative example 4 obtains vitamin K in sample2(MK-7) content is 20.18 μ g/
It is low to detect numerical value than embodiment 3 by g.Figure 17 is the chromatogram that testing sample solution carries out high-efficient liquid phase color time spectrum.
Claims (10)
1. a kind of vitamin K2(MK-7) detection method of content, it is characterised in that comprise the following processes:
A. sample pretreatment: 1. weighing quality is MSampleSample to be tested be placed in reaction vessel, be added mixed solution, be uniformly mixed
After be ultrasonically treated, be then centrifuged for taking supernatant solution；2. acid solution is added into supernatant solution, sufficiently after oscillation, after layering
The removal of lower layer's solution；3. water is added into step 2. remaining solution, sufficiently after oscillation, lower layer's solution after layering is removed；
4. the solution on collection step 3. upper layer, is spin-dried for, it is V that organic solvent constant volume, which is added, and obtains volumeSampleSample solution；
B. it prepares reference substance solution: weighing vitamin K2(MK-7) reference substance obtains concentration after organic solvent constant volume is added as CIt is right's
Reference substance solution；
C. the preparation of solution to be measured: sample solution, reference substance solution are filtered through miillpore filter respectively, obtain sample to be tested
Solution, standard reference material solution；
D. standard reference material solution is measured using high-efficient liquid phase chromatogram technology, obtains vitamin K2(MK-7) chromatographic peak
Peak area is simultaneously denoted as AIt is right, sampling volume is denoted as VIt is right；
E. it utilizes identical high-efficient liquid phase chromatogram technology condition in same step D to be measured testing sample solution, obtains dimension life
Plain K2(MK-7) peak area of chromatographic peak and it is denoted as ASample, sampling volume is denoted as VLoading；
F. vitamin K in sample to be tested is calculated according to external standard method2(MK-7) content C, calculation formula are as follows:
The unit of C is μ g/g, V in formulaSampleUnit be mL, CIt is rightUnit be μ g/mL, VIt is right、VLoadingUnit be μ L, MSampleUnit
For g.
2. a kind of vitamin K according to claim 12(MK-7) detection method of content, it is characterised in that: described to be measured
When sample is solid, first it is ground into fine powder and is pre-processed again；Sample to be tested is directly pre-processed when being liquid.
3. a kind of vitamin K according to claim 12(MK-7) detection method of content, it is characterised in that: the mixing
Solution is mixed by polar solvent and nonpolar solvent.
4. a kind of vitamin K according to claim 32(MK-7) detection method of content, it is characterised in that: the polarity
Solvent is any one of isopropanol, methanol, ethyl alcohol, acetonitrile；Nonpolar solvent is n-hexane, petroleum ether, appointing in hexamethylene
It is a kind of.
5. a kind of vitamin K according to claim 42(MK-7) detection method of content, it is characterised in that: the mixing
Solution is mixed by n-hexane and isopropanol.
6. a kind of vitamin K according to claim 52(MK-7) detection method of content, it is characterised in that: it is described just oneself
The volume ratio of alkane and isopropanol is 2~4:1.
7. a kind of vitamin K according to claim 12(MK-7) detection method of content, it is characterised in that: the acid is
Glacial acetic acid, the concentration of acid solution are 2~4mol/L.
8. a kind of vitamin K according to claim 12(MK-7) detection method of content, it is characterised in that: step A and step
Organic solvent described in rapid B is any one of methanol, ethyl alcohol, isopropanol or acetonitrile, and used organic in step A and step B
Solvent type is identical.
9. a kind of vitamin K according to claim 12(MK-7) detection method of content, it is characterised in that the efficient liquid
The condition of phase chromatography are as follows: chromatographic column is using octadecylsilane chemically bonded silica as filler or equivalent chromatographic column；Mobile phase includes second
Nitrile and methanol solution, flow rate of mobile phase are 0.8~1.5mL/min, and column temperature is 25~40 DEG C, and sample volume is 10~20 μ L, detection
Wavelength is UV, visible light 254nm.
10. a kind of vitamin K according to claim 92(MK-7) detection method of content, it is characterised in that: the stream
It is gradient elution when dynamic mutually elution, the content of acetonitrile is containing for 70~80%, methanol specially in 0~10min mobile phase
Amount is 20~30%；In 10~20min mobile phase the content of acetonitrile by 70~80% be at the uniform velocity reduced to the content of 0, methanol by
20~30% at the uniform velocity rise to 70~80%；It is methanol in 20~50min mobile phase.
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